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101.
A novel histone exchange factor, protein phosphatase 2Cgamma, mediates the exchange and dephosphorylation of H2A-H2B 下载免费PDF全文
Kimura H Takizawa N Allemand E Hori T Iborra FJ Nozaki N Muraki M Hagiwara M Krainer AR Fukagawa T Okawa K 《The Journal of cell biology》2006,175(3):389-400
In eukaryotic nuclei, DNA is wrapped around a protein octamer composed of the core histones H2A, H2B, H3, and H4, forming nucleosomes as the fundamental units of chromatin. The modification and deposition of specific histone variants play key roles in chromatin function. In this study, we established an in vitro system based on permeabilized cells that allows the assembly and exchange of histones in situ. H2A and H2B, each tagged with green fluorescent protein (GFP), are incorporated into euchromatin by exchange independently of DNA replication, and H3.1-GFP is assembled into replicated chromatin, as found in living cells. By purifying the cellular factors that assist in the incorporation of H2A-H2B, we identified protein phosphatase (PP) 2C gamma subtype (PP2Cgamma/PPM1G) as a histone chaperone that binds to and dephosphorylates H2A-H2B. The disruption of PP2Cgamma in chicken DT40 cells increased the sensitivity to caffeine, a reagent that disturbs DNA replication and damage checkpoints, suggesting the involvement of PP2Cgamma-mediated histone dephosphorylation and exchange in damage response or checkpoint recovery in higher eukaryotes. 相似文献
102.
Hanae Takatsuki Katsuya Satoh Kazunori Sano Takayuki Fuse Takehiro Nakagaki Tsuyoshi Mori Daisuke Ishibashi Ban Mihara Masaki Takao Yasushi Iwasaki Mari Yoshida Ryuichiro Atarashi Noriyuki Nishida 《PloS one》2015,10(6)
The infectious agents of the transmissible spongiform encephalopathies are composed of amyloidogenic prion protein, PrPSc. Real-time quaking-induced conversion can amplify very small amounts of PrPSc seeds in tissues/body fluids of patients or animals. Using this in vitro PrP-amyloid amplification assay, we quantitated the seeding activity of affected human brains. End-point assay using serially diluted brain homogenates of sporadic Creutzfeldt–Jakob disease patients demonstrated that 50% seeding dose (SD50) is reached approximately 1010/g brain (values varies 108.79–10.63/g). A genetic case (GSS-P102L) yielded a similar level of seeding activity in an autopsy brain sample. The range of PrPSc concentrations in the samples, determined by dot-blot assay, was 0.6–5.4 μg/g brain; therefore, we estimated that 1 SD50 unit was equivalent to 0.06–0.27 fg of PrPSc. The SD50 values of the affected brains dropped more than three orders of magnitude after autoclaving at 121°C. This new method for quantitation of human prion activity provides a new way to reduce the risk of iatrogenic prion transmission. 相似文献
103.
Investigations of rhubarb and the bark of Rhaphiolepis umbellata led to the isolation of new flavan-3-ol glucosides. Their structures were elucidated on the basis of 1H and 13C NMR analysis hydrolytic studies as (+)-catechin 5-O-β-d-glucopyranoside and (?)-catechin 7-O-β-d-glucopyranoside. 相似文献
104.
In vitro refolding process of urea-denatured microbial transglutaminase without pro-peptide sequence 总被引:5,自引:0,他引:5
Yokoyama K Kunio O Ohtsuka T Nakamura N Seguro K Ejima D 《Protein expression and purification》2002,26(2):329-335
Efficient refolding process of denatured mature microbial transglutaminase (MTG) without pro-peptide sequence was studied in the model system using urea-denatured pure MTG. Recombinant MTG, produced and purified to homogeneity according to the protocol previously reported, was denatured with 8M urea at neutral pH and rapidly diluted using various buffers. Rapid dilution with neutral pH buffers yielded low protein recovery. Reduction of protein concentration in the refolding solution did not improve protein recovery. Rapid dilution with alkaline buffers also yielded low protein recovery. However, dilution with mildly acidic buffers showed quantitative protein recovery with partial enzymatic activity, indicating that recovered protein was still arrested in the partially refolded state. Therefore, we further investigated the efficient refolding procedures of partially refolded MTG formed in the acidic buffers at low temperature (5 degrees C). Although enzymatic activity remained constant at pH 4, its hydrodynamic properties changed drastically during the 2h after the dilution. Titration of partially refolded MTG to pH 6 after 2h of incubation at pH 4.0 improved the enzymatic activity to a level comparable with that of the native enzyme. The same pH titration with incubation shorter than 2h yielded less enzymatic activity. Refolding trials performed at room temperature led to aggregation, with almost half of the activity yield obtained at 5 degrees C. We conclude that rapid dilution of urea denatured MTG under acidic pH at low temperature results in specific conformations that can then be converted to the native state by titration to physiological pH. 相似文献
105.
106.
In ants, cuticular hydrocarbons are used for nestmate recognition; they are stored in the postpharyngeal gland and shared among the individuals. Newly emerged ants have a very small quantity of hydrocarbons. We studied the ontogeny of the hydrocarbon profile in Aphaenogaster senilis. The total quantities of both cuticular and postpharyngeal gland (PPG) hydrocarbons increased with age from 0 to 20 days after emergence and then stabilised. These quantities are correlated with the development of the ovary. Under individual social isolation, cuticular hydrocarbons increased as normal, but the total quantity of PPG hydrocarbons never increased from the initial low level. This effect of social isolation on the PPG hydrocarbon level indicates the importance of hydrocarbon transfer between nestmates through the PPG and lends support to the gestalt model of nestmate recognition. To cite this article: K. Ichinose, A. Lenoir, C. R. Biologies 332 (2009). 相似文献
107.
An alternative reaction pathway of F1-ATPase suggested by rotation without 80 degrees/40 degrees substeps of a sluggish mutant at low ATP 下载免费PDF全文
F(1)-ATPase, a water-soluble portion of F(o)F(1)-ATP synthase, is a rotary motor driven by ATP hydrolysis. The central gamma-subunit rotates in the alpha(3)beta(3) cylinder by repeating four stages of rotation: ATP-binding dwell, rapid 80 degrees substep rotation, catalytic dwell, and rapid 40 degrees substep rotation. In the catalytic dwell, at least two catalytic reactions occur-cleavage of the enzyme-bound ATP and presumably release of the hydrolyzed product(s) from the enzyme-but we found that a slow ATP cleavage mutant of F(1)-ATPase from thermophilic Bacillus PS3 rotates at low ATP concentration without substeps and the catalytic dwell. Analysis indicates that in this alternative reaction pathway the two catalytic reactions occur during the preceding long ATP-binding dwell. Thus, F(1)-ATPase can operate through (at least) two competing reaction pathways, not necessarily through a simple consecutive reaction. 相似文献
108.
Saori Hata Sayaka Fujishige Yoichi Araki Naoko Kato Masahiko Araseki Masaki Nishimura Dieter Hartmann Paul Saftig Falk Fahrenholz Miyako Taniguchi Katsuya Urakami Hiroyasu Akatsu Ralph N. Martins Kazuo Yamamoto Masahiro Maeda Tohru Yamamoto Tadashi Nakaya Sam Gandy Toshiharu Suzuki 《The Journal of biological chemistry》2009,284(52):36024-36033
Alcadeins (Alcs) constitute a family of neuronal type I membrane proteins, designated Alcα, Alcβ, and Alcγ. The Alcs express in neurons dominantly and largely colocalize with the Alzheimer amyloid precursor protein (APP) in the brain. Alcs and APP show an identical function as a cargo receptor of kinesin-1. Moreover, proteolytic processing of Alc proteins appears highly similar to that of APP. We found that APP α-secretases ADAM 10 and ADAM 17 primarily cleave Alc proteins and trigger the subsequent secondary intramembranous cleavage of Alc C-terminal fragments by a presenilin-dependent γ-secretase complex, thereby generating “APP p3-like” and non-aggregative Alc peptides (p3-Alcs). We determined the complete amino acid sequence of p3-Alcα, p3-Alcβ, and p3-Alcγ, whose major species comprise 35, 37, and 31 amino acids, respectively, in human cerebrospinal fluid. We demonstrate here that variant p3-Alc C termini are modulated by FAD-linked presenilin 1 mutations increasing minor β-amyloid species Aβ42, and these mutations alter the level of minor p3-Alc species. However, the magnitudes of C-terminal alteration of p3-Alcα, p3-Alcβ, and p3-Alcγ were not equivalent, suggesting that one type of γ-secretase dysfunction does not appear in the phenotype equivalently in the cleavage of type I membrane proteins. Because these C-terminal alterations are detectable in human cerebrospinal fluid, the use of a substrate panel, including Alcs and APP, may be effective to detect γ-secretase dysfunction in the prepathogenic state of Alzheimer disease subjects. 相似文献
109.
In adaptive bone remodeling, it is believed that bone cells such as osteoblasts, osteocytes and osteoclasts can sense mechanical stimuli and modulate their remodeling activities. However, the mechanosensing mechanism by which these cells sense mechanical stimuli and transduce mechanical signals into intracellular biochemical signals is still not clearly understood. From the viewpoint of cell biomechanics, it is important to clarify the mechanical conditions under which the cellular mechanosensing mechanism is activated. The aims of this study were to evaluate a mechanical condition, that is, the local strain on the cell membrane, at the initiation point of the intracellular calcium signaling response to the applied mechanical stimulus in osteoblast-like MC3T3-E1 cells, and to investigate the effect of deformation velocity on the characteristics of the cellular response. To apply a local deformation to a single cell, a glass microneedle was directly indented to the cell and moved horizontally on the cell membrane. To observe the cellular response and the deformation of the cell membrane, intracellular calcium ions and the cell membrane were labeled using fluorescent dyes and simultaneously observed by confocal laser scanning microscopy. The strain distribution on the cell membrane attributable to the applied local deformation and the strain magnitude at the initiation point of the calcium signaling responses were analyzed using obtained fluorescence images. From two-dimensionally projected images, it was found that there is a local compressive strain at the initiation point of calcium signaling. Moreover, the cellular response revealed velocity dependence, that is, the cells seemed to respond with a higher sensitivity to a higher deformation velocity. From the viewpoint of cell biomechanics, these results provide us a fundamental understanding of the mechanosensing mechanism of osteoblast-like cells. 相似文献
110.
Nishizawa R Nishiyama T Hisaichi K Matsunaga N Minamoto C Habashita H Takaoka Y Toda M Shibayama S Tada H Sagawa K Fukushima D Maeda K Mitsuya H 《Bioorganic & medicinal chemistry letters》2007,17(3):727-731
Hydroxylated derivatives were designed and synthesized based on the information of oxidative metabolites. Compounds derived from beta-substituted (2R,3R)-2-amino-3-hydroxypropionic acid showed improved inhibitory activities against the binding of MIP-1alpha to human CCR5, compared with the non-hydroxylated derivatives and the other isomers. 相似文献